Download BD FACSAria ™ IIu SORP standard operating procedure

SAFETY MANUAL
AND
STANDARD OPERATING PROCEDURES
FOR THE
OCS Flow Cytometry Core
NEW YORK UNIVERSITY
SCHOOL OF MEDICINE
Old Bellevue C&D building, Room 645
Revised: July 2016
Version:
001
Approval Date:
Effective Date:
Approved by:
Peter Lopez
Director
Mark Olmsted
Environmental Specialist II, Environmental Health and Safety
Written by:
Michael Gregory
Technical Director
Mark Olmsted
Environmental Specialist II, Environmental Health and Safety
07/19/2016
Table of Contents
List of Key Personnel.................................................................................. 1
Important Telephone Numbers ................................................................. 1
OCS Flow Cytometry Core Laboratory Layout .......................................... 2
1.
2.
3.
4.
5.
6.
7.
Background ........................................................................................ 3
1.1 General Information ............................................................. 3
Risk to Lab Personnel ........................................................................ 3
2.1 Definition of Biohazardous Specimens ................................ 3
2.2 High-speed Cell Sorting ....................................................... 3
Containment ....................................................................................... 4
3.1 Facility Layout ...................................................................... 4
3.2 Laboratory Facilities ............................................................ 4
3.3 Biosafety Cabinets and Aerosol Management Unit ............. 4
3.4 Biohazard Labels .................................................................. 4
Facility Entry and Exit ....................................................................... 5
Training .............................................................................................. 5
Medical Requirements, Surveillance, and Responses to Exposure ... 5
6.1 Medical Requirements ......................................................... 5
6.2 Medical Precautions and Surveillance ................................. 5
6.3 Medical Response to Exposure............................................ 5
Standard Operating Procedures for the OCS
Flow Cytometry Core ........................................................................ 6
Standard Operating Procedures
FLOW-101 Standard Laboratory Practices for the OCS Core ............ 7
FLOW-102 Spill Response and Reporting ........................................14
FLOW-103 Exposure Incidents and Reporting ................................15
FLOW-104 Shipping and Receiving Infectious Substances and
On-campus Transportation of Biologicals Samples .......................17
FLOW-105 Medical and Facility Emergencies .................................18
BSL-2 Sorting User Approval ................................................................... 20
Core Facility Spill / Accident Report ........................................................21
List of Key Personnel
Name
Peter Lopez
Title
Director
Extension
x30635
Mobile
646-357-0168
Michael Gregory
Technical Director
x35097
516-641-5185
Kamilah Ryan
Asst Research Scientist x35907
917-715-5337
Yulia Chupalova
Asst Research Scientist x35907
203-570-3649
Colin Zollo
Asst Research Scientist x35907
203-430-8189
Important Telephone Numbers
Note: In an emergency, Communications can page personnel from key departments
Any Medical Center Emergency
x33911
Building Services
x35071
Communications
x37403
NYULMC Emergency Room (ER)
x35550
Bellevue Emergency Department (ED)
(212) 572-5082
Occupational Health Services (OHS)
x35120
Environmental Health and Safety (EH&S)
x35159
Mark Olmsted, Biosafety Specialist
x35161
Sayra St. Omer, Biosafety Specialist
x10593
Marta Figueroa, Associate Director
x33887
NYULMC Facilities Management
x35275
Bellevue Facilities Management
(212) 562-4779
Poison Control
(212) 764-7667
Radiation Safety
x36888
Security
x73000
1
HIV Core Laboratory Layout
2
1. Background
1.1. General Information
The BSL-2 cell-sorting laboratory is located on the 6th floor of the C&D Building of Bellevue
Hospital. A locking door restricts access to this facility. The main laboratory room contains two
class IIB re-circulating biosafety cabinets, one housing the cell sorting instrument and one for
sample preparation/tissue culture. Special instrumentation is employed in this area to contain
potential aerosols and reduce operator exposure. For more detailed information please see the
CDC web site: http://www.cdc.gov/od/ohs/biosfty/biosfty.htm
2. Risk to Lab Personnel
2.1. Definition of Biohazardous Specimens
All unfixed human and primate cell suspensions and tissues must be treated as potentially
infectious, and handled in accordance with universal precautions for blood borne pathogens (i.e.,
handle as if infected with HIV, HBV, HCV etc.). This applies to cultured cell lines as well as
primary tissue suspensions (e.g., blood, bone marrow, cells derived from solid organs). It also
applies to nonhuman cells that have been deliberately infected with known or potential human
pathogens. Although standard BSL-2 working conditions are usually acceptable for handling such
specimens, the potential of cell sorters to generate high levels of aerosolized microdroplets require
a higher level of containment. For the purposes of high speed cell sorting, specimens considered
to be potentially biohazardous include all of the following:
• Suspensions of primary human or primate cells from blood or other tissues.
• Cultured and in vitro passaged human or primate cell lines. Note that with few if any
exceptions, established human cell lines may fall into the “potentially biohazardous”
category, and therefore cannot be sorted unless specific recommendations for sorting
biohazardous specimens are followed.
• Primary cells or cell lines that have been transformed with an immortalization agent that
has the potential to transform human cells, such as Epstein-Barr virus or a potentially
oncogenic retrovirus or lentivirus.
• Any samples known to contain or have been exposed to infectious pathogens normally
handled at BSL-2 conditions. This includes agents such as viruses (HIV, HCV, HBV,
CMV, EBV, influenza, etc.), bacteria (Listeria, BCG and other attenuated mycobacteria,
staphylococci, streptococci, various Gram negative pathogens, etc.), fungi (Cryptococcus,
histoplasma, aspergillus) and protozoa (Toxoplasma, some plasmodia, cryptosporidia,
etc.).
2.2. High-speed Cell Sorting
High speed droplet based cell sorters can generate large amounts of aerosols, and recently
published standards now specify a much higher level of biocontainment for cell sorting of unfixed
human cells or other potentially biohazardous samples than have been traditionally followed. “If
aerosol containment is incomplete, the safety features of the cell sorter must be modified such
that no escape of aerosol can be detected. Alternately, sorters can be placed inside a biosafety
containment cabinet” (Ref: I Schmid et al., International Society for Analytical Cytology Biosafety
Standard for Sorting of Unfixed Cells. Cytometry Part A, 71A:414-437 (2007)).
3
Sorting of samples that represent potential toxic or infectious exposures via the aerosol route
therefore require special procedures and laboratory conditions. This is true even for agents that
are normally handled under standard BSL-2 laboratory conditions, such as primary human cell
suspensions or cell lines. The heightened concern in the case of cell sorting arises from the
possibility that cells or microorganisms may be delivered directly into the lungs of personnel in the
vicinity of a cell sorter. In theory, this could increase the risk of infection with an occult pathogen,
transfer of genetic material, sensitization to antigens or other potentially harmful effects. Although
such adverse effects have not been documented as a consequence of exposure to aerosols during
cell sorting, there is sufficient concern about this to warrant the implementation of procedures to
eliminate any excess risk to personnel.
3. Containment
3.1. Facility Layout
3.1.1. The OCS Flow Cytometry Core Laboratory is a Biosafety Level 2 (BSL 2) certified facility
that is located in Bellevue Hospital’s C&D Building, room CD645.
3.1.2. The facility is cleaned and maintained by the laboratory staff.
3.2. Laboratory Facilities
3.2.1. The laboratory has one sink for hand washing near the entrance to room CD645. An eye
wash station and emergency shower are located at the south end of the room.
3.2.1.1. The eye wash station and emergency shower are maintained and inspected by the
Bellevue Hospital Facilities Department (212-562-5106).
3.2.1.2. A second emergency shower is located in the hallway in front of room CD640.
Bottles of eye wash solution are available across the hall in room CD647 (access
code: 5603*).
3.3. Biosafety Cabinets (BSCs) and Aerosol Management Unit
3.3.1. There is one four foot Nuaire Class II BSC, and one Baker BioProtect III Class II BSC
located in the facility. The Baker BioProtect cabinet both houses the Aria Flow Cytometer
and provides extra bench space for sample preparation.
Name/ Room#
Baker BioProtect III BSC
Nuaire BSC
Room
CD645
CD645
Model
BioProtect III
Nu-425-400
BSC Serial No.
102073
14398 ST
3.3.2. Certification of BSCs - Environmental Health and Safety (EH&S) retains a vendor
(Technical Safety Services, Inc. www.techsafety.com) who certifies each BSC annually.
The certification is conducted in accordance with NSF Standard 49 and currently accepted
best practices.
3.3.3. Whisper Aerosol Management Unit –This unit is supplied by the FACSAria Cytometer
manufacturer (BD Biosciences) to remove and filter aerosols from the cytometer’s interior
compartments. The Whisper unit uses an ULPA filter that is changed every 6 months or if
airflow indicator is below 3 inches WC. There is a second Whisper unit in the lab which
can be used as a spare.
3.4. Biohazard Labels
4
All equipment used for storage of infectious agents must have biohazard labels specifying the
agent(s) stored.
4. Facility Entry and Exit
4.1. Refer to SOP FLOW-101
5. Training
Prior to being allowed independent access to or performing work independently in the facility, all
personnel will be trained by an approved lab user and must be approved by the Director of the OCS
Flow Cytometry Core.
Training will include knowledge of the Safety Manual and approved protocols, followed by
observation of a certified user performing the intended procedures. Then the trainee will work under
supervision of a certified lab user until the certified user gives approval and has successfully completed
all training requirements outlined in Form FLOW-101F (BSL-2 Sorting User Approval ). A copy is at the
end of this manual), for the new user to be certified to enter the core or work independently in the
facility.
6. Medical Requirements, Surveillance, and Responses to Exposure
6.1. Medical Requirements
Workers with a known immunodeficiency disease or who are taking immunosuppressive
medications are not permitted to work in the virus room without prior approval by the VCT Core
Laboratory Director, the Biosafety Officer/Specialist, and Occupational Health Services (OHS).
Workers with open wounds that cannot be adequately covered cannot work in the virus room.
OHS can provide medical advice to workers who are not sure whether they fall into any of these
exclusionary categories.
6.2. Medical Precautions and Surveillance
All NYULMC personnel working with patient samples are offered a Hepatitis B (HBV)
vaccination at their Employee Health Screening. Personnel who intend to work with bloodborne
pathogens or other potentially infectious materials, can obtain an HBV immunization by
contacting OHS at 212.263.5020. The Employee Health Center can also test whether an HBV
immunization is still effective.
Baseline HIV testing is required before working with biohazardous HIV-infected samples. HIV
testing can be obtained at the NYULM Occupational Health Center by contacting them at
212.263.5020. HIV testing is required subsequently in cases of accidental or suspected exposure.
Personnel are also encouraged to speak to their primary care physicians about regular HIV testing.
Non-NYULMC employees are responsible for maintenance of their HBV immunizations and for
their own HIV testing.
6.3. Medical Response to Exposure
Procedures for management of exposure due to cuts are detailed in Safety Policy 135, Bloodborne
Pathogens Exposure Control Program. These procedures apply to all NYULMC employees and as
such apply to all employees working in the OCS Flow Cytometry Core. These procedures are
stated below.
All cuts and other exposures to blood or other body fluids must be reported immediately to OHS,
or if OHS is not open at that time, to the Emergency Room (ER) in NYULMC or Bellevue
5
Hospital. The worker should also notify the OCS Flow Cytometry Core Co-Directors and the
Study Principal Investigator as soon as possible, after appropriate emergency care has been
obtained.
Follow-up treatment for all exposures in the OCS Flow Cytometry Core will be advised by,
offered by or arranged by OHS or the ER. Form FLOW-102F BSL-2 Spill/Accident Report
should be filed with the Laboratory Manager.
7. Standard Operating Procedures (SOPs) for the OCS Flow Cytometry Core
7.1. A dedicated set of SOPs and forms are to be followed and used by all personnel using the OCS
Flow Cytometry Core.
The currently approved SOPs and forms pertaining specifically to the virus laboratory are on the
following pages of this safety manual and are listed below:
Document No.
Name
SOP FLOW-101
Standard Laboratory Practices for the OCS Flow Cytometry
Core Laboratory
SOP FLOW-102
Spill Response and Reporting
SOP FLOW-103
Exposure Incidents and Reporting
SOP FLOW-104
Shipping and Receiving Infectious Substances and Oncampus Transportation of Biological Samples
SOP FLOW-105
Medical and Facility Emergencies
Form FLOW-101F
BSL-2 Sorting User Approval
Form FLOW-102F
Core Facility Spill/Accident Report
6
Standard Operating Procedures
Title: Standard Laboratory Practices for the OCS Flow Cytometry Core Laboratory
SOP#: FLOW-101
Purpose: To provide safe handling procedures and operations for all personnel working in the facility
1. Materials
Item
Lab Coats
Safety Glasses
Gloves
Manufacturer
Jackson Safety
Americare
200 Proof Ethyl Alcohol
RelyOn Multi-purpose Disinfectant
Wipes
Bleach
Kim Wipes
FACSFlow Sheath
Alcohol Swabs
Accudrop Beads
CST Beads
Buffalo ULPA Filters
GloGerm Beads
DuPont Chemical
Catalog No.
NYU Building Services
19706-002
NYU Requisition (Small 7-001)
(Medium 7-002)
(Large 7-003)
NYU Requisition
Fisher# 19-120-3881
Clorox
KimTech
BD
BD
BD
BD
Buffalo
GloGerm
Staples# CLO 02489
34155
342003
366894
345249
641319
BD# 335029
-
2. Restricted Access
2.1. Entry into the OCS Flow Cytometry Core Laboratory is restricted to authorized individuals who
have received medical clearance from OHS, have taken the Intro to Biosafety training, the OSHA
Bloodborne Pathogens self study, and reviewed the SOPs for the OCS Flow Cytometry Core
Laboratory.
2.2. NYULMC’s Biosafety Officer/Specialist and Environmental Specialists will be granted access to
conduct unannounced inspections.
2.3. Entry into the OCS Flow Cytometry Core is restricted by a keyed lock and users must always be
accompanied by a member of the Flow Core Staff unless otherwise authorized.
2.4. During sorting of potentially infectious agents, access to the laboratory will be restricted.
3. General Facility Requirements
3.1. Use of needles and other sharp instruments will not be used when biohazardous samples are
present.
3.2. All cuts in the skin must be covered with a bandage.
3.3. No food or drinks are allowed.
3.4. No open-toed shoes are to be worn in the facility.
3.5. No jewelry (other than wedding bands) is to be worn under gloves.
7
3.6. No mouth pipetting is allowed in the facility.
3.7. All samples must be labeled with name, date and specimen type with a water/alcohol resistant
marker.
3.8. Post-sort clean up should follow procedures listed in the Decontamination and Exit out of the OCS Flow
Cytometry Core sections of this SOP.
3.9. Equipment is to be decontaminated prior to maintenance
4. Reagents and Supplies
4.1. All samples that are transported to the OCS Flow Cytometry Core must be contained using
approved secondary containers. Refer to SOP FLOW-104
4.2. Unopened, non-infectous, non-toxic reagents and supplies are stored in rooms CD643 or CD647.
5. Entry to the OCS Flow Core Laboratory and Personal Protective Equipment (PPE)
5.1. All outside clothing not worn under a lab coat must be left in room CD643 (office). Bags and
anything not to be used in the Core Laboratory should be left there as well.
5.2. Wearing two pairs of gloves is required. They are disposed when overtly contaminated and
removed when work is completed or integrity is compromised. Small, medium and large gloves
are available to the left of the entrance to the room, and should be worn at all times They will be
sprayed with 70% ethanol or isopropyl alcohol as necessary and are not to be worn outside the
lab.
5.3. Lab coats are available on a coat rack behind the door and are to be worn at all times while inside
the lab. If a different size is needed, coveralls or surgical gowns can be supplied. Non-disposable
lab coats are laundered on a regular basis by NYULMC Building Services.
5.4. The laboratory door should remain closed except when entering and exiting the lab.
6. Aerosol generating procedures
All transfers of biohazardous materials from one container to another container must take place
within a BSC. Such transfers may not take place on the open bench.
All other procedures that could generate aerosols must also be conducted in a BSC. The
following are examples of these procedures:
• Mixing of samples with a pipette;
• Using high speed mixing devices like vortexers;
• Opening of centrifuge buckets; and
• Opening a package containing an infectious pathogen.
7. Use of FACSAria Flow Cytometer and Baker BioProtect III BSC
7.1.
Any biohazardous or potentially biohazardous samples run on the FACSAria have the
potential to aerosolize during instrument malfunction and contaminate the sorting chamber of
the instrument. Because of this, sort setups must be preformed in a sequence. The setup
8
procedure outlined below will insure that all necessary adjustments are made before any sample
is run on the instrument.
Step
Procedure
Comments
1
Turn on the Baker BSC blowers and
lights.
2
Turn on the FACSAria instrument
and computer.
The blowers must be on for 15 minutes before
any human samples are run.
The FACSAria needs at least 15 minutes to
warm up (30 minutes for use of the UV laser)
before step 6
Turn on Whisper Aerosol
Whisper Unit should be on for 15 minutes
Management unit, and (optionally) the
before any human samples are run.
water recirculation unit.
Refill or empty as necessary at this time. Add
Check sheath, waste and other
4
bleach to waste tank as per Decontamination
solution levels.
section of this SOP.
Run Fluidics Startup and turn stream Let stream stabilize and set gap and drop.
5
on.
Engage “sweet spot.”
6
Run CST beads for instrument QC.
Run Accudrop beads and perform
7
drop delay setup.*
Set up sort side streams for
8
appropriate collection vessels.*
9
Change necessary optical filters.*
Ensure hood hatches are closed, hood
10
pressure gauge is correct and proper
PPE is used.
11
Begin running samples/sorting.
* Steps 7, 8, and 9 must be completed before a biohazardous sample is run on the
machine. These steps require open hatches on the hood and can create aerosols.
3
7.2. In the event of a clog the stream may lose stability and generate an aerosol. In most cases, any
aerosol should be contained within the sort block and filtered out by the Whisper Aerosol
Management Unit. If the stream restarts correctly, the sort can continue.
7.2.1.
If the sort block needs to be opened, or the collection containers removed after a clog, the
operator must wait for 20 minutes before proceeding, to allow any aerosol to settle.
7.2.2. If the nozzle must be removed for cleaning, it should be treated as biohazardous. The
stream should be turned off, and the nozzled taken out of the machine. Before removal
from the hood it should be placed into a 15mL conical tube, filled with either diH2O or
EtOH, which is then capped. This tube can then be taken out of the hood and placed into
the sonicator for cleaning. The tube should not be opened again until inside the hood.
7.3. After sorting is complete the operator must wait for 5 minutes before removal of the collection
apparatus. Tubes should be capped and plates covered. All collection containers should be labeled
and wiped down with RelyOn Disinfectant Wipes before removal from the Biosafety cabinet.
9
7.4. To decontaminate the FACSAria, a tube of 70% EtOH should be run on the sample port for 5
minutes at high speed (10). Afterwards, one of two built-in automated procedures must be used.
7.4.1. If the machine is to be used again, the automated “Prepare for Aseptic Sort” procedure of
the FACSDiva Software can be initiated. This procedure will flush all lines that come in
contact with sample with 70% EtOH and disinfectant. Once complete the machine will be
flushed with clean FACSFlow Sheath solution.
7.4.2. If the machine is no longer needed for the day, the automated “Fluidics Shutdown”
procedure of the FACSDiva Software can be initiated. This will also flush the machine with
70% EtOH and disinfectant, but will not flush the machine with Sheath solution. Using
this procedure for shutdown will reduce salt buildup and help prevent future clogs.
7.5. The surfaces of the FACSAria and Baker BioProtect Cabinet should be wiped down with 70%
EtOH or RelyOn Disinfectant Wipes. Blowers should run for at least 15 minutes after cabinet has
been wiped down.
7.6. Waste from the FACSAria is pumped into a 10 liter tank on the fluidics cart below the hood. This
tank must contain enough bleach (1 liter) to render the final volume in the tank 10% bleach. The
tank is capped with a biohazard filter that allows air pressure release, but prevents aerosol from
escaping. The filter is to be changed on a monthly basis.
8. Use of Nuaire Class II Type A BSC
8.1.
Before working in the BSC, the blowers and fluorescent light are switched on, and a biohazard
bag, a spray bottle of 70% isopropyl alcohol, wipers, and pre-saturated wipes are placed in the
cabinet. The blowers must be left on for 15 minutes before use.
8.2.
All materials needed to complete the experiment are placed in the cabinet to limit the number
of times hands pass through the air barrier. Equipment is not to be placed on the intake grills
at the front of the cabinet, nor blocking the exhaust opening at the back of the cabinet.
8.3.
A biohazard bag should be present in the cabinet. Absorbent material (such as a dry cleanroom
wiper) is placed in the bottom of the biohazard bag. This bag is used for discarding solid waste
(gloves, plastic waste, pipette tips). Once the bag is full, it is closed, wiped with 70% isopropyl
alcohol and taken out of the cabinet to be collected into a larger covered waste container next
to the cabinet.
8.4.
Liquid waste should be put into a dedicated container inside the BSC with sufficient bleach to
achieve a final concentration of 10% ( 0.5% sodium hypochlorite) and allowed to react for a
minimum of 30 to 60 minutes before disposal. Wipe or spray the outside of the container with
70% ethanol or isopropyl alcohol before removing it from the cabinet. The decontaminated
liquids are then disposed of down the sink and flushed with large amounts of tap water.
8.5.
Vacuum waste flasks should contain enough bleach to result in a 10% solution. They should
never be filled more than 50%. An in-line vacuum filter must be present between the flask and
the vacuum source.
8.6.
Contaminated pipettes should be disposed of in the biohazard bags.
8.7.
Anything removed from the BSC during the work session is to be decontaminated by wiping
with 70% isopropyl alcohol while still in the BSC.
10
8.8.
At the end of each work session, culture tubes, racks and other material to be removed from
the cabinet are decontaminated by wiping with 70% isopropyl alcohol while still within the
BSC.
8.9.
The wipers used during cleaning along with the outer gloves are placed into a biohazard bag
while still within the BSC. Wipe or spray the outside of the bag with 70% isopropyl alcohol.
Place the bag into a larger covered biohazard waste container next to the cabinet.
8.10.
A fresh pair of outer gloves is donned and the hood is now wiped down completely with 70%
isopropyl alcohol.
8.11.
All tissue or cell culture related materials should be disposable whenever possible. Only
disposable plastic pipettes and plastic tubes are to be used in the facility.
9. CO2 Incubators
The following is a list of safety practices and procedures for doing work involving the use of cell
culture incubators.
NOTE: The incubator in the OCS Flow Cytometry Core is not available for tissue culture use.
The incubator is to be used only to keep samples at 37 degrees C for short periods of time just
prior to or preceding a sort.
9.1.
Flasks and culture plates shall be carried to and from the incubator using plastic secondary
containers.
9.2.
In the event of bacterial or fungal contamination in the incubators, all contents shall be moved
to a BSC. Shelves shall be wiped down with 70% isopropyl alcohol and shelves should be
sterilized in an autoclave.
9.3.
Gloves must be worn when handling cultures.
9.4.
Prior to maintenance, equipment must be decontaminated.
10. Centrifuge
The following is a list of safety practices and procedures for doing work involving the use of
centrifuges.
10.1.
Rotor buckets and lids shall be sprayed with 70% isopropyl alcohol and placed in the BSC
prior to loading.
10.2.
Samples shall be loaded into rotor/rotor buckets and sealed with the cap for the rotor bucket
while in BSC.
10.3.
After centrifuging, rotor/rotor buckets shall be moved to BSC to unload samples. Samples
shall NOT be unloaded in the open room.
10.4.
Centrifuge and rotor chambers shall be disinfected with 70% isopropyl alcohol soaked wipers
following use.
10.5.
Prior to maintenance, equipment must be decontaminated.
11
11. Decontamination
Work surfaces are to be decontaminated on completion of work, after any spill or splash, or when
switching over to a new patient or product batch.
Decontaminate as follows:
11.1. Bench tops and external equipment surfaces: Work surfaces are wiped down with 70%
ethanol or RelyOn Disinfectant Wipes.
11.2. Water baths and Sonicator: Water baths and sonicators are completely emptied of water and
wiped down with 70% ethanol or RelyOn Disinfectant Wipes.
11.3. Biosafety cabinet work surfaces: BSC work surfaces are sprayed with disinfectant cleaner
(RelyOn Multi-purpose disinfectectant cleaner or equivalent), allowing a 10 minute contact time,
followed by wiping down with 70% isopropyl alcohol to remove excess disinfectant residue.
11.4. Interior surfaces of equipment: Interior surfaces of centrifuges (including centrifuge buckets),
incubators and other large equiment are wiped down with 70% ethanol or RelyOn Disinfectant
Wipes.
11.5. Liquid Waste: Liquid biohazard waste will be decontaminated with sufficient bleach to achieve
a final concentration of 10% (0.5 % sodium hypochlorite )for a minimum of 30 to 60 minutes
and then emptied into the sink.
11.6. Other Potentially Contaminated Waste: All other potentially contaminated waste such as
disposable lab coats and gloves are collected in red bags in containers with lids. Clothing that
becomes contaminated with HIV or potentially infectious material preparations will be
decontaminated by spraying with 70% ethanol before being laundered or discarded.
11.7. All red bags containing contaminated wastes must be double-bagged and securely sealed with
tape. All sharps containers should be locked closed. Outside surfaces of both red bags and
sharps containers must be wiped down with disinfectant cleaner (RelyOn Multi-purpose
disinfectant cleaner or equivalent) before transporting out of the lab.
12. Use of Chemicals
12.1. The same practices and training requirements will apply to the use of chemicals as in all other
laboratories of NYULMC. Specifically, personnel must be current with Chemical Hygiene and
Hazardous Waste training requirements. EH&S offers training on the 2nd Thursday of each
month; contact them @ x35159 for further information.
12.2. For all chemicals used in the facility, the user must give the Laboratory Manager a corresponding
Safety Data Sheet (SDS). All personnel must be instructed as to their importance and their
location within the facility; the Laboratory Manager will be in charge of monitoring chemical
storage and use within the facility.
13. Disposal of hazardous chemicals
13.1. Hazardous chemicals will be collected in properly labeled containers in a designated area in the
lab Arrangements for the disposal of hazardous chemical waste may be made by contacting
EH&S.
13.2.
Biohazard waste cannot be discarded through the Hazardous Waste Disposal Program.
12
13.3.
Arrangements for the disposal of hazardous chemical waste that is also a biohazard may be
made by contacting EH&S.
14. Exit out of the OCS Flow Cytometry Core
14.1. All persons leaving the Core laboratory must remove PPE and wash hands before exiting.
14.2.
Solid biohazard waste (red bags and sharps containers) should be stored in designated area, as
there is no regular pickup and NYULMC Environmental Services must be notified for pickup.
14.3.
Decontaminated liquid biohazard waste should be emptied into the sink and flushed with large
amounts of tap water (Refer to 11.5 in SOP#: FLOW-101 for proper liquid decontamination practices).
14.3.1. Used liquid waste canisters should be disposed in red bag waste.
14.3.2. Secondary containers for carrying liquid waste containers are disinfected by spraying down
with 70% ethanol or isopropyl alcohol, and may be autoclaved if needed.
14.4.
Dispose gloves in a biohazard waste receptacle (red bag waste) and wash hands before exit.
13
Standard Operating Procedures
Title: Spill Response and Reporting
SOP#: FLOW-102
Purpose: To provide safe procedures for spill response in the facility
1. Materials
Item
Biohazard Bags
RelyOn disinfectant cleaner
Blue absorbent pads
Spill Kit
Manufacturer
Lab Guard
Dupont
Fisherbrand
Spill Defense
Catalog No.
19075388E
Fisher # 19-120-3881
14-206-62
25916
2. Spill Response
2.1. Spills will be decontaminated promptly by the responsible party.
2.2. Personnel in the immediate area will be alerted and access to the contaminated area (around the
spill) will be clearly marked with the biohazard floor sign and restricted.
3. Spill Clean-Up
3.1. Don a lab coat, two pairs of gloves, and eye or face protection
3.2. Use the spill kit to clean up the spill. The spill kit contains absorbent packets and pads.
3.3. Pick up any glass with tongs
3.4. Carefully cover the spill with the absorbent packets.
3.5. Taking care to avoid splashing pour a freshly prepared 1 in 10 dilution of bleach around the
edges of the spill.
3.6. Allow a 30 to 60 minutes contact time.
3.7. Use dry cleanroom wipers or the absorbent pads to wipe up the spill working from the edges
into the center.
3.8. Disinfect the spill area by spraying thoroughly with RelyOn disinfectant/cleaner, allowing a 10
minute contact time before wiping dry.
3.9. Discard waste and any contaminated PPE in a red biohazard bag.
3.10. Wash hands.
3.11. Report spills and accidents to Environmental Health & Services.
4. Reporting
Spills or accidents will be reported to the Biosafety Officer/Specialist, the Core Laboratory Manager,
and the Co-Directors of the OCS Flow Cytometry Core. Fill out the Core Facility Spill/Accident Report
Form, (Form FLOW-102F – a copy is at the end of this safety manual or can be obtained from the
Laboratory Manager) to document large spills or other potentially serious accidents.
14
Standard Operating Procedures
Title: Exposure Incidents and Reporting
SOP#: FLOW-103
Purpose: To provide safe procedures for accidental exposures
1. Emergency Procedures:
All personnel who work in the lab will be familiar with the Emergency Response Guide for New York
University Medical Center Laboratories that is posted in the lab next to the entrance. This gives basic
information on responding to fire alarms, chemical or biological spills or personal injury.
2. Exposure Incidents
Manage exposure incidents such as cuts with contaminated instruments, or splash to mucous
membranes as follows:
2.1. For cuts with contaminated instruments:
2.1.1. Stop work immediately.
2.1.2.
Remove contaminated gloves and allow the wound to bleed freely for a minute under
warm running water.
2.1.3.
Wash the wound with soap and water for 5 minutes and apply sterile gauze or a bandage,
if necessary.
2.1.4.
Remove protective lab clothing and proceed immediately to the appropriate location for
treatment and counseling.
2.2. For splashes to mucosal membranes:
2.2.1. Stop work immediately and proceed immediately to the eye wash station.
2.2.2. Rinse tissue surface with copious amounts of water. Eyes should be irrigated for at least 15
minutes.
2.2.3. Remove protective lab clothing and proceed immediately to the appropriate location for
treatment and counseling.
Appropriate Locations for Treatment and Counseling
Department
Occupational
Health Services
NYULMC
Emergency Room
Bellevue Hospital
Emergency
Department
Phone Number
Location
212-263-5020
One Park Avenue on
the 3rd Floor
212-263-5550
560 First Avenue
212-562-5082
462 First Avenue
Hours of
Operation
M-F 8:00AM5:00PM
Open 24 hours
7 days/week
Open 24 hours
7 days/week
Note: If a laboratory worker has a parenteral (e.g. percutaneous injury or contact with non-intact skin) or
mucous membrane exposure to blood, body fluid, or viral-culture material, the source material will
be identified and, if possible, tested for the presence of virus. In general, materials handled in
15
the OCS Flow Cytometry Core should be considered contaminated unless known
otherwise.
For work involving HIV-infected or potentially infected materials, the worker must be escorted
directly to the emergency room for immediate evaluation and counseling with regard to the risk of
infection. OHS offers post-exposure prophylaxis (PEP) according to the most recent
guidelines, and if deemed necessary, should begin as soon as possible, typically within
hours of exposure. Administration of PEP should not be delayed for HIV test results.
As of August 2008, the CDC recommendation is as follows:
“Use of PEP with antiretroviral medications, initiated as soon as possible after exposure and
continuing for 28 days, has been associated with a decreased risk for infection following
percutaneous exposure in health-care settings (22)…Because of the potential toxicities of
antiretroviral drugs, PEP is recommended unequivocally only for exposures to sources known
to be HIV-infected. The decision to use PEP following unknown-source exposures is to be
made on a case-by-case basis, considering the information available about the type of exposure,
known risk characteristics of the source, and prevalence in the setting concerned.” [MMWR
Aug 1, 2008 / 57(RR06); 1-19)].
The worker will be evaluated serologically for HIV and advised to report and seek medical
evaluation of any acute febrile illness that occurs within 12 weeks after the exposure. Such an
illness – particularly one characterized by fever, rash, or lymphadenopathy – may indicate recent
HIV infection. If the initial (at time of exposure) HIV test is negative, the worker should be
retested 6 weeks after the exposure and periodically thereafter (i.e., at 12 weeks and 6, 9 and 12
months after exposure). During this follow-up period exposed workers should be counseled to
follow Public Health Service recommendations for preventing transmission of HIV.
NOTE: Please note that exposure to other bloodborne pathogens or other potentially
infectious materials is discussed in detail in NYULMC’s OSHA Bloodborne
Pathogens self study.
3. Reporting
Exposure incidents must be reported immediately either in person or by phone to a OCS Flow
Cytometry Core Manager, the Co-Directors of the OCS Flow Cytometry Core, and OHS. Use Core
Facility Spill/Accident Report Form, (Form FLOW-102F – a copy is at the end of this safety manual or
can be obtained from the Laboratory Manager) to document the incident.
16
Standard Operating Procedures
Title: Shipping and Receiving Infectious Substances and On-campus Transportation of Biological
Samples
SOP#: FLOW-104
Purpose: To ensure that shipping and receiving/transportation of specimens and cultures which harbor
or are suspected of harboring pathogens is performed in a controlled and dedicated manner.
1. Training Requirements
Personnel who want to ship or receive infectious substances must be current with training
requirements.
1.1. EH&S provides the self-study packages: Introduction to Shipping Hazardous Materials and Shipping and
Receiving Biological Materials, which are available at:
http://redaf.med.nyu.edu/shipping-and-receiving-biological-materials-training
1.2. A tr aining cer tificate is issued and maintained in the EH&S Depar tment upon successful
completion of both post tests mentioned in 1.1; the cer tification is valid for two year s.
2. On-campus Transportation of Biological Samples
2.1. Materials
Item
Biohazard Bags
Hard container (cooler)
Blue absorbent pads
Manufacturer
Lab Guard
Igloo
Fisherbrand
Catalog No.
19075388E
7362
14-206-62
2.2. General Notes
2.2.1. All samples and containers must have biohazard labels.
2.2.2. Avoid crowded areas whenever possible.
2.2.3. The container should be carried directly to the intended laboratory - do not take the
container to offices, cafeterias or other public or inappropriate locations.
2.2.4. The package should be carefully inspected for signs of leakage or other contamination and,
if necessary, decontaminated before opening.
2.3. Packaging Instructions
2.3.1. Label samples. Label information must include the identity of the biological material or
agent, the universal biohazard symbol and the sending and receiving laboratory
identification (e.g., Principal Investigator name and room number).
2.3.2. Place sample in a primary container which is sealed and leak proof.
2.3.3. Place the primary container in a secondary hard case container which is easy to
decontaminate and capable of being securely closed.
2.3.4. Liquid samples should be surrounded by enough absorbent pads in the secondary
container to contain any liquids and absorb any shock during transport.
17
Standard Operating Procedures
Title: Medical and Facility Emergencies
SOP#: FLOW-105
Purpose: To provide safe procedures for handling medical and facility emergencies
1. Materials
Item
Emergency flashlight
First Aid Kit
Smart Universal Power
Supply 3000XL
Manufacturer
Sexauer
PhysiciansCare
ADP
Catalog No.
FST #456870
Staples #503995
-
2. Medical Emergencies
1.1. In case of a medical emergency, call the Medical Center’s emergency number: 33-911.
1.2. If the individual is conscious and can be moved, remove him/her immediately out of the
laboratories.
1.3. If the individual is unconscious and it will cause no further harm, the person will be immediately
removed out of the laboratories and emergency personnel will be called to perform first aid.
1.4. If the victim cannot be moved, instruct the emergency responders of hazards and protective
measures necessary in the facility.
1.5. Stay with the victim until emergency medical personnel arrive and take over.
2. Electrical Failures
2.1. In case of a power outage the operator must use his/her own best judgment to assess the
situation and act accordingly.
2.2. In case of an electrical failure, call Bellevue’s main number for Facilities: (212) 562-4779.
2.3. Loss of power to the essential containment equipment will be avoided by use of a APC Smart
UPS 3000xL battery backup system that will maintain power to the Aria Flow Cytometer,
Whisper Aerosol Containment Unit, BioSafety Cabinet, and Vacuum pump for up to two hours,
allowing proper shutdown and containment of biohazards.
2.4. If the blower fan of a BSC stops working any operator working in the BSC is required to cease
all work immediately and exit from the laboratory following the exit procedures listed in SOP
FLOW-101 for removal of protective gear.
2.4.1. The blower must be on for at least thirty minutes before work can resume.
2.5. In case of a blackout, all operators are to evacuate the facility. A rechargeable flashlight will be
available for emergency use if needed.
2.6. Exit doors are identified with glow-in-the-dark exit signs which will allow the operator to find
the exit door. Exit procedures listed in SOP FLOW-101 will be followed.
2.7. A sign should be posted on the entrance door with a notice advising persons not to enter the
18
facility.
3. Fire Emergency
3.1. In the event of a fire the laboratory worker must take the following steps:
3.1.1. If the infectious material is stored as per lab requirements, the worker removes the PPE
and exits the lab quickly as per exit procedures detailed in SOP FLOW-101, as required
when he/she leaves.
3.1.2. If research involving the infectious material is in progress, the worker will determine if the
agent can quickly be secured or whether it is quicker to destroy the material prior to leaving
the lab as outlined in the section on SOP FLOW-101 Decontamination – Liquid Waste.
3.2. After evacuating the facility on account of fire, all workers will remain at a safe distance to offer
directions to the facility and any information EH&S and/or Fire Department personnel may
request. When they or Fire Department personnel arrive on the scene, all workers will follow
their instructions.
19
FORM FLOW-101F. BSL-2 Sorting User Approval
Name: _________________________________ Date: ____________________
Date of birth: ______________
Principal Investigator: _____________________ Department: _______________
Title: __________________________________ Work phone: _______________
Home phone: ____________________________ E-mail: ___________________
____ I have completed, within the past year, the NYULMC training on bloodborne pathogens.
____ I have read (and received a copy of) the OCS Flow Cytometry Core’s SOPs and am familiar with:
§
Safe working practices, which all persons in the facility are expected to follow
§
Appropriate responses for spills in the laboratory, both within and outside of BSCs
§
Decontamination procedures
§
Procedures for medical, electrical and fire emergencies
§
The Biosafety Microbiological and Biomedical Laboratories (BMBL) manual published and
periodically updated by the Center for Diseases Control (CDC), specifically section IV –
Laboratory Biosafety Level Criteria, and section VIII E – Viral Agents (HIV/SIV).
____ In compliance with the OSHA Bloodborne Pathogens Standard, NYULMC has an HBV
vaccination program. I understand that under this program, any worker who is at risk from HBV
from occupational exposure to human blood, blood products or body fluids, or HBV
contaminated materials may receive an HBV vaccination free of charge. The HBV vaccination
program is administered by OHS.
____ I understand that the OHS and the ER are prepared to administer medications to reduce the risk
of HIV infection following a needlestick or mucous membrane exposure to HIV and it is my
responsibility to report immediately to be evaluated for such treatment in the event of a possible
exposure.
User signature: ____________________________ Date: ___________________
OCS Core Laboratory signature: ____________________ Date: ___________________
20
Form FLOW - 102F. Core Facility Spill/Accident Report
Reporting Objective:
In the process of investigating and reporting incidents the facility can determine the cause and provide
recommendations for future prevention and correction of the events that lead to the accident/spill. This
document is based on OSHA CLP 02-00-135-Recordkeeping Policies and Procedures Manual (effective12/30/2004).
If additional space is needed to complete any question for a section, please attach extra page indicating
which section is being continued.
1. Completed by (Name, Job Title):
___________________________________________________
2. Name/Job Title/Name of Principal Investigator: _______________________________________
3. Date/Time of Incident: ____________________________________________________________
4. Infectious agent/hazardous substance involved: ________________________________________
5. Where did incident happen (which area of the Core facility)? ______________________________
6. Describe circumstances that lead to incident (work being done at that time, location of spill, equipment
involved):
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
______________________________
7. Other persons in Core lab at time of incident (where were they; did they contribute to the incident?)
_____________________________________________________________
8. Duration of safety breach (time to containment): ____________________________________
9. What, if any, measures were taken to contain the safety problem?
a. Evacuation of facility ____Yes
____No
b. Who de-contaminated the spill (person or persons)? _____________________
_______________________________________________________________
10. Who was notified of the incident? When were they notified?
____________________________
_______________________________________________________________________________
_______________________________________________________________________________
____
11. List any injuries as a result of this incident:
_______________________________________________________________________________
21
_________________________________________________________________
12. What medical evaluation or treatment was sought due to the incident?
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
______
a. Emergency Room ____NYULMC _____Bellevue HC ____No
b. Occupational Health Services ____Yes____No
c. If any other medical/healthcare treatment was obtained outside the work-site, where was it
obtained:
_________________________________________________________________________
_______________________________________________________________________
13. Please suggest any future measures that could be taken to prevent a recurrence of this type of incident:
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
22